Efeito do ácido linoléico conjugado TRANS-10, CIS-12 na regulação do acúmulo de lípides e expressão gênica em embriões produzidos in vitro

Supplementation of conjugated linoleic acid trans-10, cis-10 in the culture medium, represents an important alternative to increasing the survival of embryos after cryopreservation. However the addition of culture media with CLA trans-10, cis-12 without antioxidant may increase the apoptotic rate. The aim of this study was to evaluate the effect of adding CLA trans-10, cis-12 in vitro culture of embryos without antioxidant. Bovine zygotes (n = 1,694) were divided into two treatments: (T1) control group, zygotes cultured in CR2aa medium supplemented with fetal calf serum (n = 815), (T2) zygotes cultured in CR2aa medium supplemented with fetal calf serum plus 100 µM CLA trans-10, cis-12. Embryos were evaluated for development, amount of lipids and cryotolerance. Transcripts of messenger RNA (mRNA) of selected genes were measured by real time PCR. Supplementation of CLA trans-10, cis-12 did not significantly affect the blastocyst rate (31.8% and 34.1% T1 and T2, respectively, p = 0,20) and the mRNA level of genes related to cell stress, apoptosis and de novo synthesis of fatty acid . Lipids and transcripts of the mRNA of the gene 1-acilglicerol-3-phosphate o-acyltransferase 1 enzyme related to the synthesis of triglycerides were significantly reduced in embryos cultured in the presence of CLA trans-10, cis-12 in comparison the control group. Had increased rate re-expansion of blastocysts cultured in the presence of CLA trans-10, cis-12, after thawing (34.4 and 56.3% for T1 and T2, respectively p = 0,002). This difference did not persist in the hatching rate (14.0 and 16.5% for T1 and T2, respectively, P = 0,62). These results show that the CLA trans-10, cis-12 reduces the accumulation of lipids in the embryos by reducing the levels of gene transcripts acilglicerol-1-3-phosphate oacyltransferase 1 without affecting the quality of the embryo. Additionally, this fatty acid increases the rate re-expansion, but does not improve the hatching rate.A suplementação do ácido linoléico conjugado trans-10, cis-10 no meio de cultivo, representa uma importante alternativa para aumento da sobrevivência dos embriões após a criopreservação. No entanto este isômero de CLA no cultivo in vitro sem antioxidante pode aumentar a taxa apoptótica. O objetivo do presente estudo foi avaliar o efeito da adição CLA trans-10, cis-12 no cultivo in vitro de embriões sem antioxidante. Zigotos bovinos (n = 1.694) foram divididos em dois tratamentos: (T1) grupo controle, zigotos cultivados no meio CR2aa suplementado com soro fetal (n = 815); (T2) zigotos cultivados no meio CR2aa suplementado com soro fetal mais 100 µM CLA trans-10, cis-12. Os embriões foram avaliados quanto a desenvolvimento, quantidade de lípides e criotolerância. Transcritos dos RNA mensageiros (RNAm) dos genes selecionados foram mensurados pelo Real Time PCR. Suplementação de CLA trans-10, cis-12 não afeta significativamente a taxa de blastocisto (31,8% e 34,1% T1 e T2, respectivamente, p = 0,20) e nível dos RNAm dos genes relacionados com stress celular, apoptose e síntese de novo de ácido graxo. Quantidade de lípides e transcritos do RNAm do gene 1-acilglicerol-3-fosfato oaciltransferase 1 enzima relacionado a síntese de triglicérides foram significativamente reduzidos nos embriões cultivados na presença de CLA trans-10, cis-12 em comparação com o grupo controle. Teve aumento significativo na taxa de re-expansão dos blastocistos cultivados na presença de CLA trans-10, cis-12, após o descongelamento (34.4 e 56.3% para T1 e T2, respectivamente p = 0,002). Essa diferença não persistiu na taxa de eclosão (14,0% e 16,5% para T1 e T2, respectivamente, P = 0,62). Esses resultados mostram que o CLA trans-10, cis-12 reduz o acúmulo de lípides nos embriões pela redução nos níveis dos transcritos do gene 1-acilglicerol-3-fosfato o-aciltransferase 1 sem afetar a qualidade do embrião. Adicionalmente, este ácido graxo aumenta a taxa de re-expansão, no entanto, não melhora a taxa de eclosão.FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerai

Efeito do ácido linoléico conjugado TRANS-10, CIS-12 na regulação do acúmulo de lípides e expressão gênica em embriões produzidos in vitro

A suplementação do ácido linoléico conjugado trans-10, cis-10 no meio de cultivo, representa uma importante alternativa para aumento da sobrevivência dos embriões após a criopreservação. No entanto este isômero de CLA no cultivo in vitro sem antioxidante pode aumentar a taxa apoptótica. O objetivo do presente estudo foi avaliar o efeito da adição CLA trans-10, cis-12 no cultivo in vitro de embriões sem antioxidante. Zigotos bovinos (n = 1.694) foram divididos em dois tratamentos: (T1) grupo controle, zigotos cultivados no meio CR2aa suplementado com soro fetal (n = 815); (T2) zigotos cultivados no meio CR2aa suplementado com soro fetal mais 100 µM CLA trans-10, cis-12. Os embriões foram avaliados quanto a desenvolvimento, quantidade de lípides e criotolerância. Transcritos dos RNA mensageiros (RNAm) dos genes selecionados foram mensurados pelo Real Time PCR. Suplementação de CLA trans-10, cis-12 não afeta significativamente a taxa de blastocisto (31,8% e 34,1% T1 e T2, respectivamente, p = 0,20) e nível dos RNAm dos genes relacionados com stress celular, apoptose e síntese de novo de ácido graxo. Quantidade de lípides e transcritos do RNAm do gene 1-acilglicerol-3-fosfato oaciltransferase 1 enzima relacionado a síntese de triglicérides foram significativamente reduzidos nos embriões cultivados na presença de CLA trans-10, cis-12 em comparação com o grupo controle. Teve aumento significativo na taxa de re-expansão dos blastocistos cultivados na presença de CLA trans-10, cis-12, após o descongelamento (34.4 e 56.3% para T1 e T2, respectivamente p = 0,002). Essa diferença não persistiu na taxa de eclosão (14,0% e 16,5% para T1 e T2, respectivamente, P = 0,62). Esses resultados mostram que o CLA trans-10, cis-12 reduz o acúmulo de lípides nos embriões pela redução nos níveis dos transcritos do gene 1-acilglicerol-3-fosfato o-aciltransferase 1 sem afetar a qualidade do embrião. Adicionalmente, este ácido graxo aumenta a taxa de re-expansão, no entanto, não melhora a taxa de eclosão.Supplementation of conjugated linoleic acid trans-10, cis-10 in the culture medium, represents an important alternative to increasing the survival of embryos after cryopreservation. However the addition of culture media with CLA trans-10, cis-12 without antioxidant may increase the apoptotic rate. The aim of this study was to evaluate the effect of adding CLA trans-10, cis-12 in vitro culture of embryos without antioxidant. Bovine zygotes (n = 1,694) were divided into two treatments: (T1) control group, zygotes cultured in CR2aa medium supplemented with fetal calf serum (n = 815), (T2) zygotes cultured in CR2aa medium supplemented with fetal calf serum plus 100 µM CLA trans-10, cis-12. Embryos were evaluated for development, amount of lipids and cryotolerance. Transcripts of messenger RNA (mRNA) of selected genes were measured by real time PCR. Supplementation of CLA trans-10, cis-12 did not significantly affect the blastocyst rate (31.8% and 34.1% T1 and T2, respectively, p = 0,20) and the mRNA level of genes related to cell stress, apoptosis and de novo synthesis of fatty acid . Lipids and transcripts of the mRNA of the gene 1-acilglicerol-3-phosphate o-acyltransferase 1 enzyme related to the synthesis of triglycerides were significantly reduced in embryos cultured in the presence of CLA trans-10, cis-12 in comparison the control group. Had increased rate re-expansion of blastocysts cultured in the presence of CLA trans-10, cis-12, after thawing (34.4 and 56.3% for T1 and T2, respectively p = 0,002). This difference did not persist in the hatching rate (14.0 and 16.5% for T1 and T2, respectively, P = 0,62). These results show that the CLA trans-10, cis-12 reduces the accumulation of lipids in the embryos by reducing the levels of gene transcripts acilglicerol-1-3-phosphate oacyltransferase 1 without affecting the quality of the embryo. Additionally, this fatty acid increases the rate re-expansion, but does not improve the hatching rate

Antifreeze proteins for low-temperature preservation in reproductive medicine: A systematic review over the last three decades

International audienceAntifreeze proteins (AFPs) are synthesized by diverse non-mammalian species, allowing them to survive in severely cold environments. Since the 1990s, the scientific literature reports their use for low temperature preservation of germplasm. The aim of this systematic review was to compile available scientific evidence regarding the use of AFP for low-temperature preservation of several reproductive specimens. Internet databases were consulted using the terms: "antifreeze protein" OR "AFP" OR "antifreeze glycoprotein" OR "AFGP" OR "ice-binding protein" OR "IBP" OR "thermal hysteresis protein" AND "cryopreservation". From 56 articles, 87 experiments testing AFPs in low-temperature preservation of gametes, embryos or reproductive tissues/cells were fully analyzed and outcomes were annotated. A positive outcome was considered as a statistically significant improvement on any parameter evaluated after low-temperature preservation with AFP, whereas a negative outcome included worsening of any evaluated parameter, in comparison to untreated groups or groups treated with a lower concentration of AFP. The findings indicated that research on the use of AFP as a cryoprotectant for reproductive specimens has increased markedly over the past decade. Some experiments reported both positive and negative results, which depended, on AFP concentration in the preservation media. Variation in the outcomes associated with species was also observed. Among the 66 experiments conducted in mammals, 77.3% resulted in positive, and 28.8% in negative outcomes after the use of AFP. In fishes, positive and negative outcomes were observed in 71.4% and 33.3% of 21 experiments, respectively. Most positive outcomes included preserving cell post-warming survival. The beneficial effect of AFP supports its use in cryobiological approaches used in human and veterinary medicines and animal protein industry. Moreover, combination of different AFP types, or AFP with antioxidants, or even the use of AFPbiosimilar, comprise some promising approaches to be further explored in cryopreservation

In vitro production of small ruminant embryos: latest improvements and further research

International audienceThis review presents the latest advances in and main obstacles to the application of invitro embryo production (IVEP) systems in small ruminants. This biotechnology is an extremely important tool for genetic improvement for livestock and is essential for the establishment of other biotechnologies, such as cloning and transgenesis. At present, the IVEP market is almost non-existent for small ruminants, in contrast with the trends observed in cattle. This is probably related to the lower added value of small ruminants, lower commercial demand and fewer qualified professionals interested in this area. Moreover, there are fewer research groups working on small ruminant IVEP than those working with cattle and pigs. The heterogeneity of oocytes collected from growing follicles in live females or from ovaries collected from abattoirs remains a challenge for IVEP dissemination in goats and sheep. Of note, although the logistics of oocyte collection from live small ruminant females are more complex than in the bovine, in general the IVEP outcomes, in terms of blastocyst production, are similar. We anticipate that after appropriate training and repeatable results, the commercial demand for small ruminant invitro-produced embryos may increase

Reproductive seasonality affects in vitro embryo production outcomes in adult goats

International audienceReproductive seasonality may have a considerable influence on the efficiency of assisted reproductive technologies in seasonal species. This study evaluated the effect of season on cleavage, blastocyst rates and quality of in vitro produced (IVP) goat embryos. In total, 2348 cumulus–oocyte complexes (COCs) were recovered from slaughterhouse ovaries and subjected to the same IVP system throughout 1.5 years (49 replicates). The odds ratio (OR) among seasons was calculated from values of cleavage and blastocyst rates in each season. Cleavage rate was lower (p 0.05) among seasons. In conclusion, the breeding season leads to improved oocyte developmental competence, resulting in higher cleavage and blastocyst yield, whereas embryo quality remained similar throughout the years

Criação de Mus musculus Linnaeus, 1758 como fonte alimentar para os animais do Serpentário do Ibama-JF

A proposta deste trabalho foi criar Mus musculus Linnaeus, 1758 com o objetivo de alimentar serpentes, Viperidae mantidas no serpentário do IBAMA - JF. Foram feitos primeiramente, cruzamentos com 10 matrizes doadas pelo Centro de Ensino Superior de Juiz de Fora. Após 20 dias, obteve-se 56 filhotes, que após o período de 30 dias foram agrupados separadamente por sexo. As serpentes foram levadas ao Instituto Vital Brasil não havendo necessidade de alimentação. Após dois meses, com 20 serpentes e 64 camundongos disponíveis, ocorreu a alimentação com média de três animais para cada réptil, enquanto já ocorria reprodução dos restantes. As serpentes foram separadas em caixas de transportes e alimentadas separadamente. A mesma metodologia foi aplicada durante o estudo, visto que o número de cruzamentos variava de acordo com a demanda de alimento. As serpentes são alimentadas na entrada ao Centro de Triagem de animais Silvestres (CETAS) e depois, mensalmente na proporção de 6.0% da massa corpórea das mesmas

Porcine oocyte preincubation in oviductal fluid flush before in vitro fertilization in the presence of oviductal epithelial cells improves monospermic zygote production

The present study was designed to evaluate the effect of the combination of oviduct fluid flush (OFF) and oviduct epithelial cells (OEC) in modulating the incidence of polyspermy in pigs. Therefore, for in vitro fertilization (IVF), oocyte and sperm were co-cultured in Tris-buffered medium (TBM) either supplemented with 10% OFF (OFFD group), or in the presence of a bovine OEC monolayer (OEC group), or the oocytes were exposed to OFF for 30 min before IVF (OFFB group), or in the presence of an OEC monolayer (OFFB + OEC group). Regardless of sperm concentration used (0.5, 1.5, and 4.5 × 105 cells/ml), supplementation of IVF medium with 10% OFF led to an increased (P 0.05) of the penetration rate in comparison with the control and OEC groups. When the IVF medium was supplemented with heparin, an overall increase (P < 0.05) of the final output of the IVF system in terms of zygotes with two pronuclei (2PN) was observed in the OFFD group, compared with the control and OEC groups, at a sperm concentration of 4.5 × 105 cells/ml. At this concentration, OFFB improved the monospermy rate but decreased the penetration rate, resulting in low efficiency of monospermic zygotes production. Despite this, no major effect was observed in the developmental competence of the presumed zygotes up to the blastocyst stage. The combination of OFFB with OEC improved the penetration rate, while maintaining the high monospermic rate induced by OFFB. In conclusion, the combination of treatment of oocytes by diluted OFF 30 min before IVF, followed by IVF in the presence of OEC, improved monospermic zygote production without reducing the penetration rate, when the IVF medium was supplemented with heparin.Fil: Batista, Ribrio Ivan Tavares Pereira. Institut National de la Recherche Agronomique; FranciaFil: Moro, Lucía Natalia. Institut National de la Recherche Agronomique; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Corbin, Emilie. Institut National de la Recherche Agronomique; FranciaFil: Alminana, Carmen. Institut National de la Recherche Agronomique; FranciaFil: Goncalves de Souza Fabjan, Joanna Maria. Universidade Federal do Ceara; BrasilFil: de Figueirêdo Freitas, Vicente José. Universidade Federal do Ceara; BrasilFil: Mermillod, Pascal. Institut National de la Recherche Agronomique; Franci

Combination of oviduct fluid and heparin to improve monospermic zygotes production during porcine in vitro fertilization

In vivo, the oviduct provides appropriate microenvironment conditions for monospermic fertilization and early embryo development. In addition, glycosaminoglycans such as heparin are present in the oviduct and have been shown to modulate the activity of oviduct-secreted proteins on the regulation of sperms parameters. Thus, the present study was designed to evaluate the effect of porcine oocytes exposure to oviduct fluid (OF) before in vitro fertilization (IVF; incubation of oocytes in OF for 30 minutes before IVF), during IVF (supplementation of IVF medium with 10% OF), and during IVF in combination with heparin (10% OF + 10-μg/mL heparin) on IVF parameters. Regardless of sperm concentration used (0.5, 1.5, or 4.5 × 105 cells/mL), exposure of oocytes to OF led to an increased (P 0.05) of the penetration rate in comparison with the control group. This resulted in a general increase (P 0.05) on the monospermy rate in comparison with 10% OF alone. This resulted in a general reduction (P < 0.05) in the final output of the IVF system (%), which was 33 ± 6% and 52 ± 8%, for 10% OF + heparin and 10% OF, respectively. In conclusion, the OF, used in porcine IVF, exerted a beneficial effect on oocytes by reducing the incidence of polyspermy without decreasing the penetration rate. However, the association of the OF with heparin reduced the efficiency of monospermic zygotes' production.Fil: Tavares Pereira Batista, Ribrio Ivan. Centre National de la Recherche Scientifique; Francia. Universidade Estadual Do Ceara; BrasilFil: Moro, Lucía Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Centre National de la Recherche Scientifique; FranciaFil: Corbin, Emilie. Centre National de la Recherche Scientifique; FranciaFil: Alminana, Carmen. Centre National de la Recherche Scientifique; FranciaFil: Gonçalves Souza Fabjan, Joanna Maria. Universidade Estadual Do Ceara; BrasilFil: de Figueirêdo Freitas, Vicente José. Universidade Estadual Do Ceara; BrasilFil: Mermillod, Pascal. Centre National de la Recherche Scientifique; Franci

Intrinsic quality of goat oocytes already found denuded at collection for in vitro embryo production

Remerciements : Pôle STAR; Plateforme CIRE, INRA, UMR PRC 0085, Centre Tours Val de LoireWhile cumulus cells are essential for efficient oocyte maturation, the establishment of protocols that support IVD of embryos obtained from denuded oocytes (DOC) is important for optimizing the use of reproductive biotechnologies. Thus, this study aimed to establish a protocol for IVD of goat DOC using different strategies of IVM and methods of oocyte activation. Four experiments were performed. Similar developmental competence of slaughterhouse-DOC was obtained, regardless of maturation media (complex, semi-defined or simplified). However, the ability to reach the blastocyst stage was affected by the activation method. DOC subjected to parthenogenetic activation (PA) had greater (P&lt;0.05) development capacity, compared to those undergoing IVF with average cleavage rate of 83% and 75%, blastocyst rate of 49% and 28%, and blastocysts in relation to the cleaved embryos of 59% and 38, respectively. In addition, the quality of embryos evaluated after vitrification/warming was similar between PA and IVF. Finally, we demonstrated that the co-culture of COC with DOC, increased the competence of DOC at a ratio of 1:1 and 1:9 (DOC:COC). We believe that presence of CC is not essential to the meiotic maturation, if at the time of removal of the oocyte from follicular environment, they already acquired competence to development. However, when the oocytes still need to acquire competence, the presence of CC may significantly contribute in their developmental capacity acquisition during IVM. Thus, regardless of the source, these oocytes will require longer time in IVM, contrary to what happens in the absence of CC. In conclusion, although DOC had a lower developmental potential, especially after IVF, they were able to produce blastocysts and the co-culture of DOC with COC increased this developmental capacity